Kianmehr Anvarsadat, Oladnabi Morteza, Mahrooz Abdolkarim, Ansari Javad, Mahdizadeh Rahman
Biochemistry and Metabolic Disorders Research Center, Golestan University of Medical Sciences, Gorgan, Iran; Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran.
Congenital Malformations Research Center, Golestan University of Medical Sciences, Gorgan, Iran; Department of Medical Genetics, Faculty of Advanced Medical Technologies, School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran.
Protein Expr Purif. 2018 Jun;146:1-7. doi: 10.1016/j.pep.2018.01.005.
Diaphorases are flavin-containing enzymes with potential applications in biotransfomation reactions, biosensor design and in vitro diagnostic tests. In this paper, we present recombinant expression, characterization and medium optimization of a lipoamide dehydrogenase (DLD) with NADH-dependent diaphorase activity from a Lysinibacillus sp. strain. DLD encoding sequence showed an open reading frame of 1413-bp encoding a 470 amino acid chain. Lysinibacillus sp. DLD catalyzed the NADH-dependent reduction of electron acceptors and exhibited diaphorase activity. The molecular mass of the isolated enzyme was found to be about 50 kDa, and determined to be a monomeric protein. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 7.5 and 30 °C. The K and V values were estimated to be 0.025 mM and 1.33 μmol/min, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO. By Scaling up fermentation from flask to bioreactor, enzyme activity was increased to 487.5 U/ml. This study provides data on the identification, characterization and medium optimization of a NADH-dependent diaphorase from a newly isolated Lysinibacillus sp. PAD-91.
黄递酶是含黄素的酶，在生物转化反应、生物传感器设计和体外诊断测试中具有潜在应用。在本文中，我们展示了来自芽孢杆菌属菌株的具有NADH依赖性黄递酶活性的硫辛酰胺脱氢酶（DLD）的重组表达、表征及培养基优化。DLD编码序列显示一个1413bp的开放阅读框，编码一条470个氨基酸的链。芽孢杆菌属DLD催化NADH依赖性电子受体的还原并表现出黄递酶活性。分离出的酶的分子量约为50 kDa，确定为单体蛋白。该酶催化活性的最适pH和温度分别约为pH 7.5和30℃。K和V值估计分别为0.025 mM和1.33 μmol/min。重组酶在含有10 g/L蔗糖、25 g/L酵母提取物、5 g/L NaCl和0.25 g/L MgSO的发酵培养基中最优产率。通过从摇瓶发酵放大到生物反应器，酶活性提高到487.5 U/ml。本研究提供了关于从新分离的芽孢杆菌属PAD - 91中鉴定、表征和优化NADH依赖性黄递酶的数据。
